The functional adaptability and conformational plasticity of SARS-CoV-2 spike proteins allow for the efficient modulation of complex phenotypic responses to the host receptor and antibodies. In this study, we combined atomistic simulations with mutational and perturbation-based scanning approaches to examine binding mechanisms of the SARS-CoV-2 spike proteins with three different classes of antibodies. The ensemble-based profiling of binding and allosteric propensities of the SARS-CoV-2 spike protein residues showed that these proteins can work as functionally adaptable and allosterically regulated machines. Conformational dynamics analysis revealed that binding-induced modulation of soft modes can elicit the unique protein response to different classes of antibodies. Mutational scanning heatmaps and sensitivity analysis revealed the binding energy hotspots for different classes of antibodies that are consistent with the experimental deep mutagenesis, showing that differences in the binding affinity caused by global circulating variants in spike positions K417, E484, and N501 are relatively moderate and may not fully account for the observed antibody resistance effects. Through functional dynamics analysis and perturbation-response scanning of the SARS-CoV-2 spike protein residues in the unbound form and antibody-bound forms, we examine how antibody binding can modulate allosteric propensities of spike protein residues and determine allosteric hotspots that control signal transmission and global conformational changes. These results show that residues K417, E484, and N501 targeted by circulating mutations correspond to a group of versatile allosteric centers in which small perturbations can modulate collective motions, alter the global allosteric response, and elicit binding resistance. We suggest that the SARS-CoV-2 S protein may exploit the plasticity of specific allosteric hotspots to generate escape mutants that alter the response to antibody binding without compromising the activity of the spike protein.

We developed a computational framework for comprehensive and rapid mutational scanning of binding energetics and residue interaction networks in the SARS-CoV-2 spike protein complexes. Using this approach, we integrated atomistic simulations and conformational landscaping of the SARS-CoV-2 spike protein complexes with ensemble-based mutational screening and network modeling to characterize mechanisms of structure−functional mimicry and resilience toward mutational escape by the ACE2 protein decoy and de novo designed miniprotein inhibitors. A detailed analysis of structural plasticity of the SARS-CoV-2 spike proteins obtained from atomistic simulations of conformational landscapes and sequence-based profiling of the disorder propensities revealed the intrinsically flexible regions that harbor key functional sites targeted by circulating variants. The conservation of collective dynamics in the SARS-CoV-2 spike protein complexes showed that mutational escape positions are important for modulation of functional motions and that mutational changes in these sites can alter allosteric interaction networks. Through mutational profiling of binding and allosteric propensities in the SARS-CoV-2 spike protein complexes, we identified the key binding and regulatory hotspots that collectively determine functional response and resilience of miniproteins to mutational variants. The results suggest that binding affinities and allosteric signatures of the SARS-CoV-2 complexes can be determined by dynamic crosstalk between structurally stable regulatory centers and conformationally adaptable allosteric hotspots that collectively control the resilience toward mutational escape. This may underlie a mechanism in which moderate perturbations in the mutational escape positions can induce global allosteric changes and alter functional protein response by modulating signaling in the residue interaction networks.

Structure-functional studies have recently revealed a spectrum of diverse high-affinity nanobodies with efficient neutralizing capacity against SARS-CoV-2 virus and resilience against mutational escape. In this study, we combine atomistic simulations with the ensemble-based mutational profiling of binding for the SARS-CoV-2 S-RBD complexes with a wide range of nanobodies to identify dynamic and binding affinity fingerprints and characterize the energetic determinants of nanobody-escaping mutations. Using an in silico mutational profiling approach for probing the protein stability and binding, we examine dynamics and energetics of the SARS-CoV-2 complexes with single nanobodies Nb6 and Nb20, VHH E, a pair combination VHH E + U, a biparatopic nanobody VHH VE, and a combination of the CC12.3 antibody and VHH V/W nanobodies. This study characterizes the binding energy hotspots in the SARS-CoV-2 protein and complexes with nanobodies providing a quantitative analysis of the effects of circulating variants and escaping mutations on binding that is consistent with a broad range of biochemical experiments. The results suggest that mutational escape may be controlled through structurally adaptable binding hotspots in the receptor-accessible binding epitope that are dynamically coupled to the stability centers in the distant binding epitope targeted by VHH U/V/W nanobodies. This study offers a plausible mechanism in which through cooperative dynamic changes, nanobody combinations and biparatopic nanobodies can elicit the increased binding affinity response and yield resilience to common escape mutants.

In this study, we used an integrative computational approach to examine molecular mechanisms underlying functional effects of the D614G mutation by exploring atomistic modeling of the SARS-CoV-2 spike proteins as allosteric regulatory machines. We combined coarse-grained simulations, protein stability and dynamic fluctuation communication analysis with network-based community analysis to examine structures of the native and mutant SARS-CoV-2 spike proteins in different functional states. Through distance fluctuations communication analysis, we probed stability and allosteric communication propensities of protein residues in the native and mutant SARS-CoV-2 spike proteins, providing evidence that the D614G mutation can enhance long-range signaling of the allosteric spike engine. By combining functional dynamics analysis and ensemble-based alanine scanning of the SARS-CoV-2 spike proteins we found that the D614G mutation can improve stability of the spike protein in both closed and open forms, but shifting thermodynamic preferences towards the open mutant form. Our results revealed that the D614G mutation can promote the increased number of stable communities and allosteric hub centers in the open form by reorganizing and enhancing the stability of the S1-S2 inter-domain interactions and restricting mobility of the S1 regions. This study provides atomistic-based view of allosteric communications in the SARS-CoV-2 spike proteins, suggesting that the D614G mutation can exert its primary effect through allosterically induced changes on stability and communications in the residue interaction networks.

We developed and applied a computational approach to simulate functional effects of the global circulating mutation D614G of the SARS-CoV-2 spike protein. All-atom molecular dynamics simulations are combined with deep mutational scanning and analysis of the residue interaction networks to investigate conformational landscapes and energetics of the SARS-CoV-2 spike proteins in different functional states of the D614G mutant. The results of conformational dynamics and analysis of collective motions demonstrated that the D614 site plays a key regulatory role in governing functional transitions between open and closed states. Using mutational scanning and sensitivity analysis of protein residues, we identified the stability hotspots in the SARS-CoV-2 spike structures of the mutant trimers. The results suggest that the D614G mutation can induce the increased stability of the open form acting as a driver of conformational changes, which may result in the increased exposure to the host receptor and promote infectivity of the virus. The network community analysis of the SARS-CoV-2 spike proteins showed that the D614G mutation can enhance long-range couplings between domains and strengthen the interdomain interactions in the open form, supporting the reduced shedding mechanism. This study provides the landscape-based perspective and atomistic view of the allosteric interactions and stability hotspots in the SARS-CoV-2 spike proteins, offering a useful insight into the molecular mechanisms underpinning functional effects of the global circulating mutations.